Ca2+ and ROS Crosstalk Signaling in Cardiac Mitochondria

描述（由申请人提供）：本提案的长期目标是建立一个统一的理论来描述心肌细胞中Ca 2+和活性氧（ROS）之间的串扰信号传导机制，并将这些信号传导机制转化为心脏功能的生理和病理。线粒体Ca ~（2+）和ROS在介导心肌细胞的生与死中的关键作用已被广泛认识。钙离子介导的生命与死亡之间的分离在于Ca 2+和ROS浓度之间的最佳平衡。该领域的大多数现有研究分别集中在Ca 2+或ROS稳态。然而，这两种信号通路之间的相互作用刚刚开始受到少数实验室的关注。有趣的是，有越来越多的文献表明，线粒体动力学（裂变，融合和运输）在细胞ATP，Ca 2+和ROS稳态的生理调节中起着至关重要的作用。在这个提议中，我们将研究这三个重要的组成部分（Ca 2+，ROS和线粒体裂变机制）如何沟通，以调节心脏Ca 2+和ROS串扰信号。我们将使用多学科的方法，包括细胞生物学技术（例如共聚焦显微镜）、分子生物学（例如基因转移）、生物化学（例如蛋白质印迹法），和转基因小鼠模型（例如亲环素D（CypD）敲除小鼠和mt-cpYFP转基因小鼠）来阐明ROS和Ca 2+的机制共生与线粒体分裂蛋白DLP 1和线粒体通透性转换（MPT）的独特重点。我们的中心假设是：增加的线粒体Ca 2+浓度（[Ca 2 +]m）有利于线粒体动力学向分裂的平衡，这又增加了ROS的产生。由此产生的氧化环境导致额外的线粒体Ca 2+内流。[Ca 2 +]m和ROS的增加都增强了MPT的开放概率，从而进一步增加了ROS的产生。最终，该正反馈回路被Ca 2+和ROS激活的线粒体Ca 2+流出机制（包括Na/Ca交换和MPT）抵消。这三个具体目标是：1）确定增加的[Ca 2 +]m是否促进线粒体分裂过程，然后导致ROS产生增加。2)评估含有CypD的MPT途径在[Ca 2 +] m介导的ROS产生中的作用。3)确定MPT作为线粒体快速Ca 2+流出机制的作用。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to establish a unified theory to describe the mechanisms of crosstalk signaling between Ca2+ and reactive oxygen species (ROS) in cardiac muscle cells, and to translate these signaling mechanisms to the physiology and pathology of cardiac function. The pivotal role of mitochondrial Ca2+ and ROS in mediating the life and death of cardiac muscle cells is well recognized. The separation between mitochondria-mediated life versus death resides in the finest balance between concentrations of Ca2+ and ROS. The majority of existing research in the field focuses individually either on Ca2+ or ROS homeostasis. The interaction between these two signaling pathways, however, has just begun to gain attention by a small number of laboratories. Intriguingly, there is a growing library of literature suggesting that mitochondrial dynamics (fission, fusion, and trafficking) play an essential role in the physiological regulation of cellular ATP, Ca2+, and ROS homeostasis. In this proposal, we will study how these three important components (Ca2+, ROS, and mitochondrial fission machinery) communicate to regulate cardiac Ca2+ and ROS crosstalk signaling. We will use a multidisciplinary approach encompassing techniques of cell biology (e.g. confocal microscopy), molecular biology (e.g. gene transfer), biochemistry (e.g. western blots), and transgenic mouse models (e.g. cyclophilin D (CypD) knockout mice and mt-cpYFP transgenic mice) to elucidate the mechanisms of ROS and Ca2+ symbiosis with an unique emphasis on mitochondrial fission protein DLP1 and mitochondrial permeability transition (MPT). Our central hypothesis is: an increased mitochondrial Ca2+ concentration ([Ca2+]m) favors the balance of mitochondrial dynamics towards fission that in turn increases ROS generation. The resulting oxidized environment leads to additional mitochondrial Ca2+ influx. Both the increases in [Ca2+]m and ROS enhance the opening probability of MPT that further augments ROS generation. Eventually, this positive feedback loop is counter balanced by Ca2+ and ROS activated mitochondrial Ca2+ efflux mechanisms including Na/Ca exchange and MPT. The three specific aims are: 1) To determine whether an increased [Ca2+]m promotes mitochondrial fission processes, which then lead to increase ROS generation. 2) To assess the contribution of a CypD containing MPT pathway in [Ca2+]m-mediated ROS generation. 3) To determine the role of MPT as a rapid Ca2+ efflux mechanism of mitochondria.